A mouse metallothionein (MT) 1 expression system has been constructed that renders recombinant MT as a high purity Zn-coordinated protein. Spectral changes in absorption and circular dichroism following the addition of up to 7 mol equivalents of Cd 2+ to recombinant Zn 7-MT showed that it behaves like the native protein. Exposure of Cd 7-MT to Cd 2+ resulted in further binding of these ions to the protein, although saturation was not achieved on the addition of up to 22 mol equivalents of Cd 2+ to Zn 7MT. Spectral data are compatible with a model in which the first four additional Cd 2+ ions are bound to Cd 7-MT via sulfur atoms, and indicate that no further thiol groups are involved in the binding of the excess Cd(II) over 11. Cd 2+ ions bound in excess to Cd 7-MT appear to have lower binding constants as exposure of Cd n-MT ( n > 7) species to Chelex-100 retrieved Cd 7-MT. Based on the X-ray data, the accessible surface areas of sulfur atoms in Cd 5,Zn 2-MT 2 were calculated. This led us to propose that the coordination of the first three additional Cd(II) ions to Cd 7-MT proceeds by means of SMet1OMet1, SCys7SCys13 and SCys5SCys26 pairs. Finally, comparison of the behaviour of the entire MT with that of the recombinant α MT and β MT subunits indicates that mutual influences may not be negligible.